FLAVOUR AND FRAGRANCE JOURNAL, VOL. 11,43-47 (1996)

Volatile Leaf Oils of some South-western and Southern Australian Species of the Genus . Part VIII. Subgenus Symphyomyrtus, (a) Section Bisectaria, Series Cornutae and Series Bakeranae, and (b) Section Dumaria, Unpublished Series Furfuraceae Group

C. M. Bignell and P. J. Dunlop Department of Chemistry, University of Adelaide, South Australia, 5005, Australia

J. J. Brophy Department of Organic Chemistry, University of New South Wales, Sydney, NSW, 2052, Australia

J. F. Jackson Department of Viticulture, Oenology and Horticulture, Waite Agricultural Research Institute, University of Adelaide. South Australia, 5005, Australia

The volatile leaf oils of Eucalyptus comuta Labill., E. talyuberlup D.J. Carr & S.G.M. Carr, E. newbeyi D.J. Carr & S.G.M. Carr, E. burdettiana Blakely & Steedman, E. megacornuta C.A. Gardner, E. lehmannii (Schauer) Benth., E. conferruminata D.J. Carr & S.G.M. Carr, E. bakeri Maiden, E. jutsonii Maiden, E. mannensis Boomsma subsp. mannensis, E. leptocalyx Blakely, E. scyphocalyx (F. Muell, ex Benth.) Maiden & Blakely, E. platycorys Maiden & Blakely isolated by vacuum distillation, were analysed by GC-MS. Most species contained a-pinene (1.1-46%), 1,&cineole (0.2-86. I%), aromadendrene/terpinen-4-ol (0-9.5%) and tram-pinocarveol (0.1-13.3%) as principal leaf oil components.

KEY WORDS Labill.; Eucalyptus talyuberlup D.J. Carr & S.G.M. Can; Eucalyptus newbeyi D.J. Carr & S.G.M.Carr; Blakely & Steedman; C.A. Gardner; Eucalyptus lehmannii (Schauer) Benth.; Eucalyptus conferrum’nata D.J. Carr & S.G.M. Carr; Eucalyptus bakeri Maiden; Eucalyptus jutsonii Maiden; Eucalyptus mannemis Booomsma subsp. mannemis; Eucalyptus leptocalyx Blakely; Eucalyptus scyphocalyx (F. Muell. ex Benth.) Maiden & Blakely; Eucalyptus platycorys Maiden & Blakely; ; leaf essential oil composition; torquatone; mono- and sesquiterpenoids; GC-MS

INTRODUCTION and D.A. Kleinig).* All the species of these three Continuing our investigation of the volatile leaf series are native to the southern regions of West- oils of indigenous Australian eucalypts’-’ we em Australia except for E. mannensis Boomsma have examined the leaf oils of the seven species subsp. mannensh which is also found in Central belonging to series Comutae and the three species Australia and E. bakeri Maiden which is native to belonging to series Bakeranae, section Bisectaria south-eastem Queensland. Locations of individual of the Eucalyptus subgenus Symphyomyrtus; the species are listed in Table 1. To our knowledge three species belonging to series Furfuraceae, sec- only the oils of E. corn~ta,~E. lehm~nnU,~*~~ E. tion Dumaria of the subgenus Symphyomyrtus bakeri,”*” and E. mannensis” have been pre- were also examined (see system M.I.H. Brooker viously investigated. ccc0882-57341%/010043-05 Received 15 March 1995 0 1996 by John Wiley & Sons, Ltd. Accepted I9 June 1995 44 C. M. BIGNELL ETAL.

EXPERIMENTAL Table 1. Oil yields from the Eucalyptus species' Species and locality Oil yield Samples of clean, mature leaves were picked from wt% over ten sites on single trees and, after drying and (dry weight) freezing with liquid nitrogen, were reduced to a fine powder using a stainless steel Waring blender SERIES Cornutae Benth. Eucalyptus cornura Labill. 0.40 (Model no. SSllO). The dry powder was then Wittunga arboretum, South Australia vacuum distilled so that the leaf oil condensed on E. ralyuberlup D.J. Cam & S.G.M. Carr tr to a gold-plated copper rod maintained at approx- Waite arboretum, South Australia imately -75°C. Complete details of this proce- E. newbeyi D.J. Carr & S.G.M. Carr tr Wittunga arboretum, South Australia dure have been published previously. l4 All oils E. burdettiana Blakely & Steedman tr obtained were colourless to pale yellow liquids Wittunga arboretum, South Australia which floated on water. Table 1 lists the oil yields E. megacornura C.A. Gardner tr (wt%, dry weight) for the 13 species studied. Valley Orchids arboretum, South Australia Analytical gas chromatography (GC) was E. lehmannii (Schauer) Benth. 2.04 Wittunga arboretum, South Australia carried out on a Shimadzu GC6 AMP gas E. conferrwninata D.J. Cam & S.G.M. Carr 0.74 chromatograph. A glass SCOT column of SPlOOO Waite arboretum, South Australia (85m X 0.5mm) which was programmed from SERIES Bakeranae Chippendale 65°C to 225°C at 3"C/min was used with helium E. bakeri Maiden 2.44 carrier gas. The GC integrations of the peaks Wittunga arboretum, South Australia were performed on a SMAD electronic integra- E. jutsonii Maiden 2.24 tor. GC analyses were also performed with a Monarto arboretum, South Australia E. mannensis Boomsma subsp. mannemis 2.52 HP5890 Series I1 unit operated in conjunction Waite arboretum, South Australia with a HP3396 Series I1 integrator. The 'on- SERIES Furfuraceae BdeP column' injection technique was used with a SGE E. leptocalyx Blakely 0.79 BP20 capillary column of (25 m x 0.33 mm i.d., Monarto arboretum, South Australia and film thickness 0.5~).The carrier gas was E. scyphocalyx (F. Muell ex Benth.) 2.93 hydrogen with an inlet pressure of 25 kPa: the flow Maiden & Blakely rate was 2.0cm3/min. The oven was programmed Waite arboretum, South Australia E. platycorys Maiden & Blakely 4.00 to rise from 80°C to 220°C at 5"/min, and the inlet Waite arboretum, South Australia temperature set to 83°C and increased at the same rate as the column. Using these conditions a 2.04 a The specimens for these species were authenticated by Mr sample of 0.2% solution of oil in purified dry ether M. 1. H. Brooker, Australian National Herbarium, or Dean Nicolle, Valley Orchids, South Australia. essentially all the components were recorded by ' An unpublished Series. the integrator in 31 minutes. GC-MS was per- formed on a VG Quattro mass spectrometer present in almost all oil samples; when this was operating at 70 eV ionization energy. The GC col- not the case a sufficient amount was added to the umn in this case was DB-Wax (60m x 0.32mm). oil solution to obtain that reference point.* The Compounds were identified by their GC retention normalized retention times of the column were indices to known compounds and by comparison identified with oil components analysed previously of their mass spectra either with known com- by GC-MS for over 75 Eucalyptus species: some pounds or published spe~tra.'~-'' of these results have been published. '-' Only three of the species (E. conferruminutu, All GC analyses were performed in duplicate E. leptocalyx and E. bakeri) were analysed with and the retention times and percentage compo- GC-MS. The oil components of the rest were sitions of each component averaged. Duplicate identified using normalized retention times. For times were discarded if they differed by more this purpose the column was calibrated by assuming than one second. Components which contributed times for three markers, 1,&cineole, octadecane less than 0.06% to the final analyses were not (OD added to the ether) and torquatone. The raw considered (an arbitrary but practical decision). retention times were first normalized to 525 s for OD, and times before and after adjusted by OD *A sample of pure torquatone was kindly supplied by Dr assuming linearity and using 99 s for cineole and Emilio Ghisalberti, Chemistry Department, University of 997 s for torquatone. Torquatone was found to be . VOLATILE LEAF OILS OF EUCALYPTUS SPECIES 45

RESULTS AND DISCUSSION Acknowledgements-The authors thank Mr Ian Brooker, Australian National Herbarium, and Dean Nimlle, Valley Freshly isolated oils obtained by vacuum distilla- Orchids, South Australia, for identifying the species and help ful discussions. We are grateful to Professor Harold Woolhouse, tion of powdered leaves from single trees were analysed by GC and by GC-MS. The results for *Private communiqut from Dr R. D. Trengove, Chemistry seven species of series Cornutae, three species of Department, University of Western Australia, Director of Bakeranae and three species of Furfuraceae are the Waite Agricultural Research Institute, and Dr Jennifer Gardner, Curator of the Waite Arboretum, for their interest listed in Table 2; only those components with in this study. This work was supported in part by a grant from concentrations greater than 0.05% are reported. the Australian Research Council to P.J.D. and J.F.J., and a The principal components in the oils were the grant from the Australian Council for International Agri- monoterpenes a-pinene (1.1-46%), P-pinene cultural Research (ACIAR) to J.J.B. (0-9.4%), l,&cineole (0.2-86%) and p-cymene (O.2-6%). There were smaller amounts of limo- nene (0.2-4.3%), y-terpinene (0-15%) and a- terpineol (0.2-5.8%). Apart from 1,&cineole, REFERENCES the main oxygenated monoterpenes detected were pinocarvone (0.1-5%) and trans-pinocarveol 1. Part I. C. M. Bignell, P. J. Dunlop, J. J. Brophy and J. (O.1-13%). F. Jackson, Flavour and Fragr., 9, 113 (1994). The principal sesquiterpenes encountered in 2. Part 11. C. M. Bignell, P. J. Dunlop. J. J. Brophy and J. F. Jackson, Flavour and Fragr. J., 9, 167 (1994). the species of these three series were the hydro- 3. Part 111. C. M. Bignell, P. J. Dunlop, J. J. Brophy and J. carbons aromadendrene (0-9.5%) and allo- F. Jackson, Flavour and Fragr. J., 9, 309 (1994). aromadendrene (0-1.7%), and the related alco- 4. Part IV. C. M. Bignell, P. J. Dunlop, J. J. Brophy and J. hols, globulol (0.1-lo%), viridiflorol (0.1-1.5%) F. Jackson, Flavour and Fragr. J., 10, 85 (1995). and spathulenol (0-4.8%), as well as y-eudesmol 5. Part V. C. M. Bignell, P. J. Dunlop, J. J. Brophy and J. F. Jackson, Flavour and Fragr. J., 10, 313 (1995). (O-O.8%), a-eudesmol(O-2.1%) and P-eudesmol 6. Part VI. C. M. Bignell, P. J. Dunlop, J. J. Brophy and J. (0-3.6%). The aromatic ketone torquatone (2,4,- F.Jackson, Flavour and Fragr. J., 10,359 (1995). 6-trimethoxy-3,5-dimethyl- 1-(3-methylbutyroyl)- 7. Part VII. C. M. Bignell, P. J. Dunlop, J. J. Brophy and J. benzene) was detected (tr-17%) in all of the F. Jackson. Flavour and Fragr. J., 11, 35 (1996). 8. M. I. H. Brooker and D. A. Kleinig, A Field Guide fo the thirteen species. Eucalypts, Vol. 2, lnkata Press, Melbourne (1990). Our oil analyses agreed reasonably well with the 9. R. T. Baker and H. G. Smith, A Research on the results of the more recent measurements published Eucalyprs, Especially in Regard to their Essenfial Oils, 2nd in the literature: 1".11.13 in general many more com- edn, Government Printer, Sydney (1920). ponent compounds were detected and most of 10. E. Dellacassa. P. Mentndez, P. Moyna and E. Soler. Flavour and Fragr. J., 5, 91 (1990). them identified except in the case of E. newbeyi. 11. J. J. Brophy and D. J. Boland in Treesfor the Tropics, ed. In parts V16 and VII' of this series we indicated D. J. Boland, p. 205, Australian Centre for International that hydrodistillation may cause some com- Agricultural Research, Canberra (1989). ponents in the essential oils to decompose. In 12. A. R. Penfold, J. Proc. Roy. SOC. N.S.W., 61, 179 (1927). those two papers bicyclogermacrene were shown 13. J. J. Brophy and E. V. Lassak, Proc. Roy. SOC. N.S.W., to yield other products when hydrodistillation was 119, 103 (1986). used to generate oil from the leaves of E. sparsa; 14. R. B. Inman, P. J. Dunlop and J. F. Jackson in Modern this problem does not occur when oil is obtained Methods of Analysis New Series, Vol. 12, ed. H. F. from those leaves using supercritical C02extrac- Linskens and J. F. Jackson, p. 201, Springer-Verlag. Heidelberg (1991). tion. * More recently we have found a similar 15. S. R. Heller and G. W. A. Milne, EPAINIH Mass Spec- problem when studying the species E. cladocalyx. tral Data Base, U.S. Government Printing Office, Because of the above experimental facts we do Washington, DC (1978, 1980, 1983). not expect that our analyses, obtained with oil 16. E. Stenhagen, S. Abrahamsson and F. W. McLafferty, Registry of Mass Spectral Data. John Wiley, New York extracted by vacuum distillation, should show (1974). close agreement with the data in the literature 17. A. A. Swigar and R. M. Silverstein, Monoterpenes, obtained by hydrodistillation. Aldrich, Milwaukee, WI (1981). 1 a-Pinene 39.09 1.48 10.68 45.76 27.16 23.44 10.06 2.37 1.10 19.95 19.64 30.30 2 Camphene 0.26 - - - - 0.12 0.06 - - 0.09 - - 3 p-Pinene - - 0.01 - 0.29 0.14 9.36 2.72 0.19 0.33 0.48 0.49 4 Sabinene 0.64 - - 1.03 - - - 0.77 0.59 - - - 5 Myrcene - - 0.13 0.26 - 0.25 0.40 0.06 - 0.30 0.21 6 a-Phellandrene ------0.19 - - - 0.71 0 7 Isobutyl isovalerate 0.17- - 0.11 0.19 0.16 0.10 - - - -3 8 Limonene 4.19 0.34 1.13 1.57 4.27 2.97 2.20 0.32 0.88 1.46 2.16 2.34 9 p-Phellandrene - - - - 1.44 - - - 1.87 - - - E 10 1,8-Cineole 0.90 0.24 0.72 0.23 32.39 22.08 62.95 86.10 79.78 37.84 44.30 37.49 9 11 p-tram-Ocimene 1.43 - 0.16 0.20 ------rrn 12 y-Terpinene 2.53 - - 0.14 15.06 0.10 0.30 - - 0.16 0.17 r 13 p-Cymene 5.88 0.16 0.39 0.20 4.09 0.31 3.46 1.04- 4.01 0.69 0.18 1.45 3 14 Terpinolene 1.81 1.93 0.62 2.66 - - - - - 0.12 0.19 0.08 15 a-Cubebene ------0.07 0.19 - - - - !- 16 a-Copaene 0.15 0.14 0.10 0.10 0.06 0.16 - - 0.16 0.26 0.07 17 a-Campholenic aldehyde 1.54 1.93 2.71 0.67 - 0.17 0.08 - - - 0.07 - 18 a-Gurjunene 0.08 0.23 0.65 0.33 0.06 - - - 0.32 0.44 19 Linalol ------0.20- 0.23- - 20 from-p-Menth-2en-1-01 - 0.05 - - - - 0.26 0.10 - - 21 Pinocamone 1.54 1.54 5.04 0.45 0.11 1.16- 0.56- 0.16 0.11 3.23- 0.56 0.31 22 p-Gurjunene 0.88 - - - - 0.73 0.38 - - 0.17- - - 23 p-Elemene 0.29 0.32 0.40 0.67 - 0.11 - - - 0.26 0.25 0.47 24 f3-Cqophyllene 0.64 - 2.56 0.16 0.36 0.62 - - 0.23 0.65 1.86 25 (AromadendrenelTerpinen-4-01) 2.65 4.20 3.86 8.46 1.71 2.33 1.63 - 1.16- 9.45 5.02 10.01 26 a-Bulnesene - 0.23 0.20 0.32 - 0.11 0.06 - - 0.25 - 0.24 27 Myrtenal ------0.08 0.03 0.12 - 28 cis-p-Menth-2-en-1-01 - 0.21 - - - - - 0.10 0.06 - - 29 C15H24 ------0.37 0.12 - - - - 30 allo-Aromadendrene 0.73 1.42 1.48 1.69 0.35 0.62 0.49 0.09 - 1.31 0.70 1.28 31 trans-Pinocameol 2.91 6.15 13.30 1.73 0.45 4.49 0.87 0.11 0.19 9.13 1.11 0.94 32 Humulene ------0.18 33 6-Terpineol - 0.62 - 0.08 - - 0.12 0.10 - - - - 34 Cryptone ------0.13 0.10 2.45 0.10 0.06 - 35 cb-Piperitol - 0.65 0.24 - - - - - 0.06 - - 36 ClSH24 - - 0.19 - 0.43- 0.17 0.09 - - - 0.13 0.21 37 Viridiflorene - - 0.14 0.42 0.17 - 0.18 - - - - - 0.17 38 a-Terpineol 1.16 5.26 5.84 3.07 2.02 1.24 3.51 0.88 0.18 0.25 0.30 0.51 0.36 39 Borneo1 - - - 1.60 0.56 0.31 0.93 0.18 - - 0.19 - 0.06 40 Verbenone - - 0.16 0.11 - - - 0.W - 0.14 - - - 41 PSelinene - 0.82 - - - 0.12 0.07 - - 0.34 - 0.21 42 a-Selinene - 0.74 0.66 - 0.23 0.07 - - 0.22 - 43 A Muurolene - - 0.27 ------0.11 0.14 0.16 44 Bicyclogermacrene 0.19- 0.92 1.78 7.96 2.52 0.13 0.95 - - - 0.14 0.83 0.79 45 Carvone 0.19 - - 0.36 - - - - - 0.25 0.06 - - 46 from-Piperitol 0.07 - - - - 0.12 0.10 - - 0.09 - 0.12 - 47 8-Cadinene - 0.48 - 0.63 0.42 0.16 0.14 0.09 - - 0.07 0.25 0.18 48 C15H22 0.15 - - 0.13 - - - - 0.09 - - - - 49 Myrtenol 0.17 0.08 0.48 0.39 0.57 - 0.15 0.61 0.22 - 0.22 - - 50 Cadina-l.4-diene ------0.07 0.35 51 trum-pMentha-1 (7) ,8-dien-2-ol 1.14 - - - - - 0.29 0.29 - - 0.53 0.08 - 52 Cuminal ------0.22 - - - 53 Calamenene - 0.10 ------54 trons-p-Mentha-l,8-dien-601 - 0.14 0.29 - 1.43 - - 0.14 0.06 - 0.16 0.06 - 55 p-Cymen-8-01 0.14 0.15 0.49 0.31 0.20 0.12 0.19 0.13 - 0.17 0.09 - 0.10 56 Geraniol 0.08 - 0.11 0.10 ------57 cis-p-Mentha-1,8-dien-64 - - 0.09 0.11 - - 0.08 - - 0.08 58 ClsHsO 1.97 - 0.38 ------59 cis-p-Mentha-l(7) .8-dien-2-ol - 0.14 - 0.50 0.06 0.21 0.38 0.11 - - 0.45 0.14 0.06 60 Calacorene - 0.28 - 0.76 0.20 0.13 0.19 - - - 0.08 - 0.07 61 CuHxO ------0.11 - - 0.09 0.08 - 62 Caryophyllene oxide 0.17 0.11 0.77 1.04 0.12 0.29 0.12 - - 0.21 0.12 0.10 0.15 63 8-Phenylethylpropionate 0.07 0.60 2.49 0.43 0.26 0.09 ------64 C15H260 - - - 0.22 0.20 - 0.09 - - 0.20 0.13 0.08 0.11 65 CisHxO 0.07 0.21 0.68 0.58 0.74 0.11 0.34 0.15 - 0.12 0.65 0.46 0.62 66 Ci,Hz60 0.35 0.99 0.64 0.34 0.07 0.13 0.06 - - 0.14 0.17 0.12 67 CI5Hz6O - 0.17 0.55 0.33 0.48 - 0.24 0.09 - - 0.18 0.10 0.13 68 C15H260 - 0.09 1.07 0.26 0.14 - 0.07 ------69 Globulol 0.24- 3.84 9.70 7.13 4.63 0.39 1.58 0.79 0.06 0.23 2.86 1.74 2.45 70 Viridiflorol 0.12 0.62 1.51 1.19 1.08 0.13 0.62 0.30 0.17 0.07 0.67 0.59 0.60 71 CisH26O - 0.08 0.61 0.23 0.20 - - 0.09 - 0.06 0.06 0.16 0.10 72 Cd260 0.13 0.29 0.80 0.49 0.73 0.06 0.32 0.11 - 0.56 0.33 0.29 0.32 73 C1sHx0 0.07 0.44 1.12 0.60 1.04 0.07 0.50 0.22 - 0.07 0.32 0.30 0.35 74 Spathulenol 0.30 0.23 4.30 4.84 0.81 0.74 0.48 - - 0.24 0.41 0.22 0.41 75 y-Eudesmol 0.08 - 0.45 0.26 0.21 0.13 0.79 - - 0.06 0.20 0.58 - 76 8-Cadinol - 0.18 0.44 0.21 0.15 - 0.32 0.10 0.31 - 77 C15H260 - - 0.60 - 0.27 ------77 C15H260 ------0.07 ------79 a-Eudesmol 0.45 0.34 0.72 0.50 0.60 0.62 1.36 - - 0.21 0.34 2.07 0.06 80 8-Eudesmol 0.15 0.83 1.24 0.96 1.04 0.50 3.46 - 0.15 0.40 1.95 3.60 0.05 81 Ci5H240 ------0.06 - - - 82 C15H240 0.10 0.29 0.74 0.21 0.16 0.08 0.13 - - - - 0.10 - 83 Torquatone 1.80 1.47 2.70 1.60 1.72 0.16 16.98 - 0.25 0.08 0.33 1.88 - Total percentages 89.6 86.7 63.8 83.9 89.8 94.8 94.7 98.6- 98.3 95.8 96.5 97.1 91.8

A dash in a column indicates apercentage Composition between 0 and 0.05%.